F. Rob Jackson

Altered circadian pacemaker functions and cyclic AMP rhythms in the drosophila learning mutant dunce

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. We have examined pacemaker resetting and free-running activity rhythms in Drosophila dunce (dnc) and DC0 mutants, which identify a cAMP specific phosphodiesterase and the catalytic subunit of cAMP-dependent protein kinase, respectively. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Interestingly, however, light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Furthermore, we demonstrate the presence of circadian rhythms in cAMP content in head tissues and show that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity.

Drosophila GABAergic Systems: Sequence and Expression of Glutamic Acid Decarboxylase

Abstract: A mammalian glutamic acid decarboxylase (GAD) cDNA probe has been utilized to isolate Drosophila cDNA clones that represent a genomic locus in chromosome region 64A. Deletion analysis indicates that this chromosomal locus encodes an enzymatically active GAD protein. The in vitro translation of cRNA representing a Drosophila cDNA clone yields a 57‐kDa protein that can be immunoprecipitated by an anti‐GAD antiserum. A GAD‐immunoreactive protein of the same size can also be detected in Drosophila head extracts. The nucleotide sequence derived from two overlapping Drosophila cDNA clones predicts a 57,759‐dalton protein composed of 510 residues that is 53% identical to mammalian GAD. Sequence comparisons of mammalian and Drosophila GAD identify two highly conserved regions (≥70% identity), one of which encompasses a putative cofactor‐binding domain. Transcriptional analyses show that expression of the Drosophila Gad gene commences early in embryonic development (4–8 h) and continues in all later developmental stages. A 3.1‐kb class of mRNA is detected throughout embryogenesis, in all three larval stages, in pupae, and in adults. This transcript class has a widespread distribution in the adult CNS. A smaller 2.6‐kb transcript is expressed in a developmentally regulated manner; it is detected only in embryos and pupae.

Regulation of a specific circadian clock output pathway by lark, a putative RNA-binding protein with repressor activity†

An endogenous clock within the Drosophila brain regulates circadian rhythms in adult eclosion and locomotor activity. Although molecular elements of the Drosophila circadian clock have been well characterized, little is known about the clock output pathways that mediate the control of rhythmic events. Previous genetic analysis indicates that a gene known as lark encodes an element of the clock output pathway regulating adult eclosion. We now present evidence that lark encodes a novel member of the RNA recognition motif (RRM) class of RNA-binding proteins. Similar to other members of this protein superfamily, lark contains two copies of a bipartite consensus RNA-binding motif. Unlike any other RRM family member, however, lark protein also contains a distinct class of nucleic acid binding motif, a retroviral-type zinc finger, that is present in the nucleocapsid protein of retroviruses and in several eukaryotic proteins. In contrast to identified clock elements, lark mRNA does not exhibit diurnal fluctuations in abundance in late pupae or in adult heads. Thus rhythmic transcription of the gene does not contribute to the temporal regulation of eclosion by lark protein. Gene dosage experiments show that decreased or increased lark product, respectively, leads to an early or late eclosion phenotype, indicating that the protein negatively regulates the eclosion process. It is postulated that lark is required for the posttranscriptional repression of genes encoding other elements of this clock output pathway.

Period protein from the giant silkmoth antheraea pernyi functions as a circadian clock element in drosophila melanogaster

Homologs of the Drosophila clock gene per have recently been cloned in Lepidopteran and Blattarian insect species. To assess the extent to which clock mechanisms are conserved among phylogenetically distant species, we determined whether PER protein from the silkmoth Antheraea pernyi can function in the Drosophila circadian timing system. When expressed in transgenic Drosophila, the silkmoth PER protein is detected in the expected neural cell types, with diurnal changes in abundance that are similar to those observed in wild-type fruitflies. Behavioral analysis demonstrates that the silkmoth protein can serve as a molecular element of the Drosophila clock system; expression of the protein shortens circadian period in a dose-dependent manner and restores pacemaker functions to arrhythmic per0 mutants. This comparative study also suggests that the involvement of PER in different aspects of circadian timing, such as period determination, strength of rhythmicity, and clock out-put, requires distinct molecular interactions.

The tip-E Mutation of Drosophila Decreases Saxitoxin Binding and Interacts with Other Mutations Affecting Nerve Membrane Excitability

A recessive temperature-sensitive paralytic mutation, tip-E, is associated with reduced binding of [3H]saxitoxin to voltage-sensitive sodium channels in membranes from adult Drosophila heads. There is a decrease of 30-40% in the number of [3H]saxitoxin-binding sites per mg protein (Bmax), but the dissociation constant (Kd) for [3H]saxitoxin binding is normal in the remaining population of binding sites. This decrease is not due to a general hypotrophy of neural tissue since the number of alpha-bungarotoxin binding sites is normal in tip-E mutants. Although saxitoxin binding is reduced in vitro, pharmacological experiments suggest that tip-E mutants have close to the wild-type number of sodium channels in vivo. This suggestion is supported by the observation that at permissive temperatures tip-E only marginally suppresses a mutation which causes enhanced membrane excitability. However, even at permissive temperatures tip-E interacts synergistically with mutations that decrease membrane excitability. In this case, the double mutants exhibit reduced viability and/or longevity. We postulate that either the structure of sodium channels or their microenvironment is altered in tip-E mutants resulting in an increased liability of binding sites in vitro.

Prokaryotic and eukaryotic pyridoxal-dependent decarboxylases are homologous

SummaryA database search has revealed significant and extensive sequence similarities among prokaryotic and eukaryotic pyridoxal phosphate (PLP)-dependent decarboxylases, includingDrosophila glutamic acid decarboxylase (GAD) and bacterial histidine decarboxylase (HDC). Based on these findings, the sequences of seven PLP-dependent decarboxylases from five different organisms have been aligned to derive a consensus sequence for this family of enzymes. In addition, quantitative methods have been employed to calculate the relative evolutionary distances between pairs of the decarboxylases comprising this family. The multiple sequence analysis together with the quantitative results strongly suggest an ancient and common origin for all PLP-dependent decarboxylases. This analysis also indicates that prokaryotic and eukaryotic HDC activities evolved independently. Finally, a sensitive search algorithm (PROFILE) was unable to detect additional members of this decarboxylase family in protein sequence databases.

Structural organization and developmental expression of the protein isoaspartyl methyltransferase gene from Drosophila melanogaster

A protein carboxyl methyltransferase activity (PCMT) with a specificity for age-damaged protein D-aspartyl and L-isoaspartyl residues (E.C. 2.1.1.77) has been identified and cloned in Drosophila. The Drosophila gene was localized by chromosome in-situ hybridization to region 83AB of the third chromosome. The methyltransferase coding sequence is distributed among four exons within a 1.4-kb segment of the genome; it predicts a polypeptide of 226 amino acids that is 55% identical to the mouse enzyme. When expressed in bacteria, the Drosophila protein exhibits PCMT activity. A single 1.4-kb Pcmt transcript is detected in RNA preparations from embryos, larvae, pupae and adults. The abundance of the transcript, which is lowest in larvae and highest in adults, parallels the specific activity of the enzyme measured in extracts from the same developmental stages. It has been proposed that the PCMT initiates the repair of structurally damaged cellular proteins. The constitutive expression of PCMT and the relatively high level of expression in postmitotic adult cells suggest that PCMT activity is required through development, but acquires additional significance in aging tissues.

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.

Transcriptional Organization of a Drosophila Glutamic Acid Decarboxylase Gene

Abstract: We previously described the sequence and expression pattern of a Drosophila mRNA (Gad) that encodes the major soluble form of glutamic acid decarboxylase (GAD). We now report the transcriptional organization of the Drosophila Gad gene. Based on a combination of DNA sequence, RNase protection, primer extension, and polymerase chain reaction analyses, we conclude that the transcription unit for a 3.1‐kb Gad mRNA is composed of eight exons that span an ∼17‐kb genomic interval. By this analysis, the site of Gad transcript initiation overlaps with a recognition sequence that confers binding of the zeste transcription factor to other promoter elements. We emphasize that our analysis of the Gad transcription unit provides no evidence for alternative RNA splicing as a mechanism for the generation of GAD isoforms. Thus, the several GAD‐immunoreactive proteins (putative GAD isoforms) that can be detected in Drosophila extracts are probably encoded by distinct genes.